1. Field of the Invention
Carotenoids play a vital role in human health and nutrition. Physiological functions of carotenoids include a critical role in stem cell differentiation and concomitant growth and development. Carotenoids also play a substantial role in eye physiology, both in photo transduction and in helping to protect the retina from damage due to actinic light (for example, UV radiation). Carotenoids naturally accumulate in the macula lutea, the central region of the retina that contains the highest concentration of cone cells, which are responsible for high resolution vision. Other physiological functions include a vital immune system role through T and B cell differentiation and proliferation in response to pathogens. Carotenoids can also be efficient free radical scavengers and contribute to the oxidative stability of membranes and tissues.
Despite the importance of carotenoids in human health, mammalian cells do not synthesize most of these compounds. Mammals require dietary precursors for in vivo synthesis of key regulatory carotenoids such as retinoids. Hence, carotenoids must be present in the diet for proper health to be maintained.
Carotenoids are terpenoid compounds that are naturally synthesized in plants, microalgae, cyanobacteria, and well as some fungi and bacteria. Carotenoids can include xanthophylls, the molecules of which contain oxygen (for example, lutein and zeaxanthin) and carotenes, which are unoxygenated molecules such as α-carotene, β-carotene and lycopene.
Many different dietary carotenoids play a significant health role, among them the α-, β-, and γ-carotenes and β-cryptoxanthin, which can be converted to vitamin A in mammals. Vitamin A plays an essential role in photoreceptor function, in which light is converted to electrical signals. Since vitamin A levels depend upon an adequate intake of dietary carotenoids, poor diet can result in vitamin A deficiency with an accompanying suite of adverse health consequences.
In developing nations, particularly in sub-Saharan Africa, vitamin A deficiency can result in night blindness, and in severe cases, complete blindness. Up to half a million children in the developing world go blind each year from vitamin A deficiency, which is followed by death within a year for about half the children. Night blindness is also common in pregnant and nursing women who are vitamin A deficient. Vitamin A deficiency also correlates with a higher incidence of malaria, higher fatalities from infectious diseases, and growth stunting in infants and children. Since vitamin A levels derive from dietary intake of carotenoid compounds, there is a compelling need to improve the quantity, quality and availability of carotenoids in diet.
Although animals do not synthesize carotenoids, some microbial species have carotenoid synthesis pathways. Carotenoid synthesis genes have also been successfully transferred into microbial species that do not naturally produce the compounds. Microbial production of carotenoids, whether natural or transgenic, can be efficient enough to be used in industrial production. Many commercial facilities around the world culture microaglae microalgae for the purpose of harvesting carotenoids.
While the capacity of microbes to commercially produce carotenoids as bulk chemicals has been explored, with an extensive number of patents granted, the potential for using gut microbiota to synthesize carotenoids in situ as dietary inputs has not been grasped until very recently. While the intestinal bacterium Escherichia coli has been experimentally transformed with plasmid vectors carrying genes coding for carotenoids, in most cases, E. coli was used simply as a laboratory model to ascertain gene function and to study the biochemical pathways involved in compound synthesis, including the MVA and MEP pathways. The use of carotenoid-producing Escherichia strains to correct nutritional deficiencies or to boost systemic carotenoid or vitamin A levels has not been patented, as far as the investigator has been able to determine. This also holds true for other gut microbiota, such as lactic acid bacteria, as well for free-living carotenoid synthesizing microbes which might be adapted for probiotic use.
Lactic acid bacteria have an ancient history of safe human use in foods and food products, being the active microbial agents in the making of dairy products such as yogurts and cheeses, as well as numerous vegetable fermentations, such as sauerkraut, kimchi, olives and pickled cucumbers. Lactic acid bacteria carry the US-FDA designation of “generally regarded as safe,” or GRAS. Lactic acid bacteria ingested in fermented foods or in capsule form can colonize the digestive tract, often resulting in improvement in digestive function, alleviation of diarrhea and other intestinal ailments and inflammations, improved immune function and may also contribute to the inhibition of some cancers. Because of the important health role played by lactic acid bacteria and other beneficial gut microbiota such as bifidobacteria, as well as certain yeasts and non-lactic acid bacteria, including E. coli, they are collectively known as “probiotics.” The UN Food and Agriculture Organization defines probiotics as “Live microorganisms which when administered in adequate amounts confer a health benefit on the host.”
Probiotics therefore constitute an excellent platform for synthesis and delivery of carotenoids, with the goal of boosting systemic carotenoid levels in the host and alleviating vitamin A deficiencies. This can be achieved either by selection of microbial strains naturally synthesizing carotenoids, or employing transgenic probiotic organisms that synthesize carotenes using genetic material taken from other microbes, or genetic material originally isolated from plants or other sources. In this conception, carotenoid-synthesizing probiotics can be delivered in the form of capsules containing live organisms, or in cultured foods such yogurts, cheeses and soy products, or in wine or beer or fermented cereals. The boost in carotenoid levels in the host can come from two sources: either the in situ synthesis of the compounds by probiotic organisms' colonizing the host's digestive tract which have been selected or transformed for carotenoid synthesis, or by accumulation of carotenoids in a food product, such as yogurt or cheese. The overall health benefits accruing to the host are multiple: the beneficial action of the organisms themselves in the host digestive system, the in situ synthesis of carotenoid compounds by these microbes, and the higher carotenoid content of the ingested food products.
2. Description of Prior Art
Inventions claiming use of carotenoid biosynthesis by microbes have almost exclusively focused on production of extractable chemicals for food, feed and pharmaceutical use as an end goal (Li et al. 2012; Das et al. 2007; Johnson & Shroeder, 1995; U.S. Pat. Nos. 4,439,629; 5,466,599; 5,591,343; 6,783,951). There is an extensive body of literature describing the genes coding for carotenoid synthases and the genetic pathways involved (Takaichi, 2011; Cunningham & Gantt, 2007; Schmidt-Dannert. 2000). Consequently, there are many patents for genes coding for particular steps in carotenoid synthesis pathways, which often claim novelty based on the biological source of the gene (U.S. Pat. Nos. 6,929,928; 7,064,196; 7,070,952; 7,202,067; 7,582,451; 7,585,659).
There is also an extensive body of literature on probiotic organisms and the health benefits accruing from consumption of these organisms (Sears, 2005; Sanders & Klaenhammer, 2001). Numerous patents exist defining a particular strain of probiotic organism along with its particular physiological or bioactive effects (U.S. Pat. Nos. 8,071,086; 8,168,171; 8,187,590; 8,192,978). Similarly, the use of particular strains and blends of different strains or species has been the subject of many patents (U.S. Pat. Nos. 6,060,050; 8,101,170; 8,124,070). The use of live microbes in food products or dietary supplements that also contain vitamins as separately added components has also been patented (U.S. Pat. No. 8,097,281). A U.S. patent has been granted for probiotic Bifodobacterium strains specifically selected for folic acid production, with intended human use (U.S. Pat. No. 8,168,171).
Foods and beverages using fermentative microorganisms have been known since antiquity. Many processes for producing yogurts, cheeses, sauerkraut, pickles, beers, wines and numerous other products have been published or patented. Some of the microorganisms traditionally used in fermentations naturally synthesize carotenoids, such as Lactobacillus plantarum, although carotenoid production is seldom taken into account or even mentioned in descriptions of the microbial species present (Yu et al. 2012). A study of the probiotic potential of 98 strains of L. plantarum isolated from cheeses did not cite carotenoid production at all (Zago et al. 2011). Nevertheless, the genetic alteration of L. plantarum to convert the native C30 carotenoids into C40 carotenoids with known benefit to humans was been proposed; but with the object of industrial production of carotenoids (Garrido-Fernandez, 2010). Similarly, yeasts naturally synthesizing carotenoids occur widely in nature and have also been modified to produce high yields of useful carotenoids, again with the goal of commercial carotenoid production (Lin et al. 2012; Moline et al. 2012; Frengova & Beshkova, 2009). Techniques for the culture of Rhodotorula yeasts on dairy whey ultrafiltrate, either alone or in combination with symbiotic bacteria, have been developed, but with the goal of industrial production (Frengova et al. 2004).
The use of live microbes specifically to produce or accumulate carotenoids in food or beverage products or in vivo in the human digestive tract for human health purposes has not been the subject of a patent, insofar as the inventor is aware. The first formal publication of this concept was an announcement by the Gates Foundation in November, 2011, awarding Grand Challenges Explorations grants to two projects seeking to alleviate vitamin A deficiency in developing nations via probiotic carotenoid synthesis (Gates Foundation GCE Round 7). The inventor of the present invention; who was a recipient of the above grant, filed a provisional patent application prior to the award announcement date. The full presentation of the invention is given below.